Biomedical subjects

Moustapha Kassem

Publications and source records attributed to Moustapha Kassem.

At least 19 recordsLinked to original sources

PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗

PubMed2006

dlk1/FA1 regulates the function of human bone marrow mesenchymal stem cells by modulating gene expression of pro-inflammatory cytokines and immune response-related factors.

dlk1/FA1 (delta-like 1/fetal antigen-1) is a member of the epidermal growth factor-like homeotic protein family whose expression is known to modulate the differentiation signals of mesenchymal and hematopoietic stem cells in bone marrow. We have demonstrated previously that Dlk1 can maintain the human bone marrow mesenchymal stem cells (hMSC) in an undifferentiated state. To identify the molecular mechanisms underlying these effects, we compared the basal gene expression pattern in Dlk1-overexpressing hMSC cells (hMSC-dlk1) versus control hMSC (negative for Dlk1 expression) by using Affymetrix HG-U133A microarrays. In response to Dlk1 expression, 128 genes were significantly up-regulated (with >2-fold; p < 0.001), and 24% of these genes were annotated as immune response-related factors, including pro-inflammatory cytokines, in addition to factors involved in the complement system, apoptosis, and cell adhesion. Also, addition of purified FA1 to hMSC up-regulated the same factors in a dose-dependent manner. As biological consequences of up-regulating these immune response-related factors, we showed that the inhibitory effects of dlk1 on osteoblast and adipocyte differentiation of hMSC are associated with Dlk1-induced cytokine expression. Furthermore, Dlk1 promoted B cell proliferation, synergized the immune response effects of the bacterial endotoxin lipopolysaccharide on hMSC, and led to marked transactivation of the NF-kappaB. Our data suggest a new role for Dlk1 in regulating the multiple biological functions of hMSC by influencing the composition of their microenvironment "niche." Our findings also demonstrate a role for Dlk1 in mediating the immune response.

Adipocytes↗

PubMed2006

Effects of bone protein extract on human mesenchymal stem cells proliferation and differentiation.

Since its osteoinductive capacity has been established, demineralized bone matrix is considered a suitable alternative to bone autograft in the healing of osseous defects. The mechanisms of bone formation induction are still not fully understood. In this study we assessed the effects of a dispersion of bovine bone extract COLLOSS (BPE) with regard to proliferation and differentiation of a human mesenchymal stem cell line overexpressing human telomerase reverse transcriptase (hMSC-TERT). Proliferation rate was determined by (3)H-thymidine incorporation. The differentiation of hMSC-TERT cells to osteoblastic cells was assessed by means of measuring alkaline phosphatase activity and collagen synthesis in vitro. Both undifferentiated and osteoblast-differentiated hMSC-TERT cells were investigated for response to BPE. The metabolic responses to BPE were compared to unstimulated cells and cells stimulated with bovine collagen (COL). Undifferentiated hMSC-TERT cells responded to BPE with increased proliferation and decreased alkaline phosphatase activity. Osteoblastic differentiated hMSC-TERT cells had a diminished proliferative response and an increased alkaline phosphatase activity and collagen synthesis. Our study demonstrated significant metabolic effects of BPE on hMSC-TERT cells, which were highly dependant on the differentiated state of the cells.

Alkaline Phosphatase↗

PubMed2006

Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds.

Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.

Biocompatible Materials↗

PubMed2006

Smooth muscle cells in atherosclerosis originate from the local vessel wall and not circulating progenitor cells in ApoE knockout mice.

OBJECTIVE: Recent studies of bone marrow (BM)-transplanted apoE knockout (apoE-/-) mice have concluded that a substantial fraction of smooth muscle cells (SMCs) in atherosclerosis arise from circulating progenitor cells of hematopoietic origin. This pathway, however, remains controversial. In the present study, we reexamined the origin of plaque SMCs in apoE-/- mice by a series of BM transplantations and in a novel model of atherosclerosis induced in surgically transferred arterial segments. METHODS AND RESULTS: We analyzed plaques in lethally irradiated apoE-/- mice reconstituted with sex-mismatched BM cells from eGFP+ apoE-/- mice, which ubiquitously express enhanced green fluorescent protein (eGFP), but did not find a single SMC of donor BM origin among approximately 10,000 SMC profiles analyzed. We then transplanted arterial segments between eGFP+ apoE-/- and apoE-/- mice (isotransplantation except for the eGFP transgene) and induced atherosclerosis focally within the graft by a recently invented collar technique. No eGFP+ SMCs were found in plaques that developed in apoE-/- artery segments grafted into eGFP+ apoE-/- mice. Concordantly, 96% of SMCs were eGFP+ in plaques induced in eGFP+ apoE-/- artery segments grafted into apoE-/- mice. CONCLUSIONS: These experiments show that SMCs in atherosclerotic plaques are exclusively derived from the local vessel wall in apoE-/- mice.

Animals↗

PubMed2006

Playing with bone and fat.

The relationship between bone and fat formation within the bone marrow microenvironment is complex and remains an area of active investigation. Classical in vitro and in vivo studies strongly support an inverse relationship between the commitment of bone marrow-derived mesenchymal stem cells or stromal cells to the adipocyte and osteoblast lineage pathways. In this review, we focus on the recent literature exploring the mechanisms underlying these differentiation events and discuss their implications relevant to osteoporosis and regenerative medicine.

Adipocytes↗

PubMed2006

Sensitivity of fibroblast growth factor 23 measurements in tumor-induced osteomalacia.

CONTEXT: Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome of hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum 1,25-dihydryxyvitamin-D concentration, myopathy, and osteomalacia. Fibroblast growth factor 23 (FGF23) is a phosphaturic protein overexpressed in tumors that cause TIO and is, at least partly, responsible for the manifestations of TIO. OBJECTIVE: The objective of this study was to determine the sensitivity of FGF23 measurements in TIO. DESIGN: FGF23 concentrations were measured on stored samples with three ELISAs. SETTING: This study was conducted at subspecialty referral centers. PATIENTS: Twenty-two patients with suspected TIO, 13 with confirmed tumors, were studied. INTERVENTIONS: There were no interventions in this study. MAIN OUTCOME MEASURE: FGF23 concentration was the main outcome measure of this study. RESULTS: Elevated FGF23 concentrations were detected using the Immunotopics C-terminal assay in 16 of 22 TIO patients (for a sensitivity of 73%), the Immunotopics Intact assay in five of 22 patients (sensitivity, 23%), and the Kainos Intact assay in 19 of 22 patients (sensitivity, 86%). In the 13 patients with confirmed tumors, the sensitivity was higher with all assays: 92% for the Immunotopics C-terminal assay, 38% for the Immunotopics Intact assay, and 100% for the Kainos assay. CONCLUSION: The Kainos Intact assay was the most sensitive, followed by the Immunotopics C-terminal assay. The findings of normal FGF23 concentrations in some patients with TIO may indicate that FGF 23 is not responsible for the hypophosphatemia in these patients or that FGF23 secretion by some tumors is partially responsive to serum phosphate. Normal FGF23 concentrations should be interpreted in relation to the serum phosphate and 1,25-dihydryxyvitamin-D concentrations.

Adolescent↗

PubMed2006

Inhibition of osteoblast differentiation but not adipocyte differentiation of mesenchymal stem cells by sera obtained from aged females.

Aging is associated with decreased osteoblast-mediated bone formation leading to bone loss and increased risk for osteoporotic fractures. However, the cellular mechanisms responsible for impaired osteoblast functions are poorly understood. In the present study, we hypothesized that changes in bone microenvironment composition with aging are responsible for impaired osteoprogenitor cell recruitment and differentiation. As a model for bone microenvironment, we examined the effects of sera obtained from young (age 20-30 year old [yo], n=20) and old (70-84 yo, n=19) healthy female donors on cell proliferation and differentiation capacity into osteoblasts and adipocytes of human mesenchymal stem cells (hMSC). Cell proliferation rate determined by counting cell number was similar when the cells were cultured in the presence of media containing 5% sera from old or from young donors. Similarly, the number of adipocytes and levels of adipocytic gene expression was similar in cultures incubated with sera from young or old donors. We observed decreased osteoblastic gene expression in hMSC cultured either in pooled or individual sera of old donors compared to sera from young donors: core binding factor/runt-related binding factor 2 (Cbfa1/Runx2) 46%+/-2% (P<0.05), alkaline phosphatase (ALP) 45%+/-2% (P<0.05), collagen type I (Col-I) 50%+/-1% (P<0.05), and osteocalcin 65%+/-3% (P<0.05). This down-regulation of the mRNA was accompanied by reduced ALP enzyme activity by 25%+/-1% (P<0.01), immunocytochemical staining for osteoblastic markers: ALP, Col-I, and bone sialoprotein (BSP) as well as reduced in vitro mineralization as determined by Alizarin red staining. In conclusion, age-related changes in the serum composition and possibly hMSC microenvironment may contribute to the impaired osteoblast functions with aging. The factors mediating these changes remain to be determined.

Adipocytes↗

PubMed2006

Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro.

Bone marrow stromal cells (BMSCs) and other cell populations derived from mesenchymal precursors are developed for cell-based therapeutic strategies and undergo cellular stress during ex vivo procedures. Reactive oxygen species (ROS) of cellular and environmental origin are involved in redox signaling, cumulative cell damage, senescence, and tumor development. Selenium-dependent (glutathione peroxidases [GPxs] and thioredoxin reductases [TrxRs]) and selenium-independent (superoxide dismutases [SODs] and catalase [CAT]) enzyme systems regulate cellular ROS steady state levels. SODs process superoxide anion to hydrogen peroxide, which is subsequently neutralized by GPx and CAT; TrxR neutralizes other ROS, such as peroxinitrite. Primary BMSCs and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT) express GPx1-3, TrxR1, TrxR2, SOD1, SOD2, and CAT. We show here that in standard cell cultures (5%-10% fetal calf serum, 5-10 nM selenite), the activity of antioxidative selenoenzymes is impaired in hMSC-TERT and BMSCs. Under these conditions, the superoxide anion processing enzyme SOD1 is not sufficiently stimulated by an ROS load. Resulting oxidative stress favors generation of micronuclei in BMSCs. Supplementation of selenite (100 nM) restores basal GPx and TrxR activity, rescues basal and ROS-stimulated SOD1 mRNA expression and activity, and reduces ROS accumulation in hMSC-TERT and micronuclei generation in BMSCs. In conclusion, BMSCs in routine cell culture have low antioxidative capacity and are subjected to oxidative stress, as indicated by the generation of micronuclei. Selenite supplementation of BMSC cultures appears to be an important countermeasure to restore their antioxidative capacity and to reduce cell damage in the context of tissue engineering and transplantation procedures.

Base Sequence↗

PubMed2006

Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E.

Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins associated with the translation initiation eIF4F complex that may affect complex formation were identified and quantitated under distinct growth conditions. Site-specific phosphorylation of eIF4E and eIF4G and elevated levels of eIF4G:eIF4E complexes in phorbol ester treated HEK293 cells, and in serum-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP complexes, for functions related to RNP assembly, remodeling and/or transition from active translation to mRNA degradation. Our results demonstrate that our quantitative proteomic strategy can be applied to the identification and quantitation of protein complex components in human cells grown under different conditions.

Amino Acid Sequence↗

PubMed2006

Stem cells: potential therapy for age-related diseases.

Aging is associated with a progressive failing of tissues and organs of the human body leading to a large number of age-related diseases. Regenerative medicine is an emerging clinical discipline that aims to employ cellular medicines (normal cells, ex vivo expanded cells, or tissue-engineered organs) to restore the functions of damaged or defective tissues and organs and thus to "rejuvenate" the failing aging body. One of the most important sources for cellular medicine is embryonic and adult (somatic) stem cells (SSCs). One example of SCCs with enormous clinical potential is the mesenchymal stem cells (MSCs) that are present in the bone marrow and are able to differentiate into cell types such as osteoblasts, chondrocytes, endothelial cells, and probably also neuron-like cells. Because of the ease of their isolation and their extensive differentiation potential, MSCs are among the first stem cell types to be introduced in the clinic. Some recent studies have demonstrated the possible use of MSCs in systemic transplantation for systemic diseases, local implantation for local tissue defects, as a vehicle for genes in gene therapy protocols, or to generate transplantable tissues and organs in tissue-engineering protocols. However, several challenges confront the use of these cells in the clinic, ranging from biological challenges (e.g., how to isolate a homogenous populations of the cells with specific criteria from the bone marrow and how to expand them ex vivo without affecting their differentiation potential) to biotechnological challenges (e.g., how to develop easy methods for quality control of the cellular-based products). While it is expected that cellular medicines will decrease the burden of several age-related diseases, it is not clear whether they can change the course of the aging process itself and thus prolong human life.

Aging↗

PubMed2006

Heat shock-induced enhancement of osteoblastic differentiation of hTERT-immortalized mesenchymal stem cells.

Heat shock (HS)-induced stress response in human cells results in a variety of biological effects and is known to induce the transcription of heat-shock proteins, which help the cells to cope with different kinds of stress. We have studied the effects of HS on the differentiation of human mesenchymal stem cells (hMSCs) into osteoblastic cells. As a model for hMSCs we used a telomerase-immortalized hMSC line designated hMSC-TERT. Cells were exposed to 1 h HS at 41 degrees C, 42.5 degrees C, or 44 degrees C prior to incubation in a medium containing either 10(-8) M 1alpha,25-dihydroxy-vitamin-D(3) (calcitriol) or 10(-8) M calcitriol, 50 microg/mL L-ascorbic acid, and 10 mM beta-glycerophosphate followed by an analysis of induction of osteoblast differentiation and the formation of mineralized matrix, respectively. Our results indicate that the exposure of cells to mild heat stress enhances the extent of differentiation of hMSCs by 12% to 42%. These effects are an expression of the phenomenon of mild stress-induced hormesis.

Adult↗

PubMed2005

Resveratrol inhibits myeloma cell growth, prevents osteoclast formation, and promotes osteoblast differentiation.

Multiple myeloma is characterized by the accumulation of clonal malignant plasma cells in the bone marrow, which stimulates bone destruction by osteoclasts and reduces bone formation by osteoblasts. In turn, the changed bone microenvironment sustains survival of myeloma cells. Therefore, a challenge for treating multiple myeloma is discovering drugs targeting not only myeloma cells but also osteoclasts and osteoblasts. Because resveratrol (trans-3,4',5-trihydroxystilbene) is reported to display antitumor activities on a variety of human cancer cells, we investigated the effects of this natural compound on myeloma and bone cells. We found that resveratrol reduces dose-dependently the growth of myeloma cell lines (RPMI 8226 and OPM-2) by a mechanism involving cell apoptosis. In cultures of human primary monocytes, resveratrol inhibits dose-dependently receptor activator of nuclear factor-kappaB (NF-kappaB) ligand-induced formation of tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells, TRACP activity in the medium, up-regulation of cathepsin K gene expression, and bone resorption. These inhibitions are associated with a down-regulation of RANK expression at both mRNA and cell surface protein levels and a decrease of NFATc1 stimulation and NF-kappaB nuclear translocation, whereas the gene expression of c-fms, CD14, and CD11a is up-regulated. Finally, resveratrol promotes dose-dependently the expression of osteoblast markers like osteocalcin and osteopontin in human bone marrow mesenchymal stem cells (hMSC-TERT) and stimulates their response to 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. Moreover, resveratrol up-regulates dose-dependently the expression of 1,25(OH)2D3 nuclear receptor. Taken together, these results suggest that resveratrol or its derivatives deserve attention as potential drugs for treating multiple myeloma.

Apoptosis↗

PubMed2005

Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation.

Closely related signals often lead to very different cellular outcomes. We found that the differentiation of human mesenchymal stem cells into bone-forming cells is stimulated by epidermal growth factor (EGF) but not platelet-derived growth factor (PDGF). We used mass spectrometry-based proteomics to comprehensively compare proteins that were tyrosine phosphorylated in response to EGF and PDGF and their associated partners. More than 90% of these signaling proteins were used by both ligands, whereas the phosphatidylinositol 3-kinase (PI3K) pathway was exclusively activated by PDGF, implicating it as a possible control point. Indeed, chemical inhibition of PI3K in PDGF-stimulated cells removed the differential effect of the two growth factors, bestowing full differentiation effect onto PDGF. Thus, quantitative proteomics can directly compare entire signaling networks and discover critical differences capable of changing cell fate.

Cell Differentiation↗

PubMed2005

Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells.

Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation or if the stem cell origin allowed most cells to behave as cancer stem cells. Cultures of the hMSC-TERT20 strain at population doubling 440 were highly clonogenic (94%). From 110 single-cell clones expanded by 20 population doublings, 6 underwent detailed comparison. Like the parental population, each clone had approximately 1.2 days doubling time with loss of contact inhibition. All retained 1,25-(OH)(2) vitamin D(3)-induced expression of osteoblastic markers: collagen type I, alkaline phosphatase, and osteocalcin. All shared INK4a/ARF gene locus deletion and epigenetic silencing of the DBCCR1 tumor suppressor gene. Despite in vitro commonality, only four of six clones shared the growth kinetics and 100% tumorigenicity of the parental population. In contrast, one clone consistently formed latent tumors and the other established tumors with only 30% penetrance. Changing the in vitro microenvironment to mimic in vivo growth aspects revealed concordant clonal heterogeneity. Latent tumor growth correlated with extracellular matrix entrapment of multicellular spheroids and high procollagen type III expression. Poor tumorigenicity correlated with in vitro serum dependence and high p27(Kip1) expression. Aggressive tumorigenicity correlated with good viability plus capillary morphogenesis on serum starvation and high cyclin D1 expression. Thus, hMSC-TERT20 clones represent cancer stem cells with hierarchical tumorigenicity, providing new models to explore the stem cell hypothesis for cancer.

Adult↗

PubMed2005

Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation.

Human bone marrow mesenchymal stem cells (hMSC) represent a population of stem cells that are capable of differentiation into multiple lineages. However, these cells exhibit senescence-associated growth arrest and phenotypic changes during long-term in vitro culture. We have recently demonstrated that overexpression of human telomerase reverse transcriptase (hTERT) in hMSC reconstitutes telomerase activity and extends life span of the cells [Nat. Biotechnol. 20 (2002) 592]. In the present study, we have performed extensive characterization of three independent cell lines derived from the parental hMSC-TERT cell line based on different plating densities during expansion in culture: 1:2 (hMSC-TERT2), 1:4 (hMSC-TERT4), and 1:20 (hMSC-TERT20). The 3 cell lines exhibited differences in morphology and growth rates but they all maintained the characteristics of self-renewing stem cells and the ability to differentiate into multiple mesoderm-type cell lineages: osteoblasts, adipocytes, chondrocytes, and endothelial-like cells over a 3-year period in culture. Also, surface marker studies using flow cytometry showed a pattern similar to that known from normal hMSC. Thus, telomerization of hMSC by hTERT overexpression maintains the stem cell phenotype of hMSC and it may be a useful tool for obtaining enough number of cells with a stable phenotype for mechanistic studies of cell differentiation and for tissue engineering protocols.

Bone Marrow Cells↗

PubMed2005

Differential expression profiling of membrane proteins by quantitative proteomics in a human mesenchymal stem cell line undergoing osteoblast differentiation.

One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely useful for analyzing complex protein expression patterns and, when applied quantitatively, can be used to resolve subtle differences between samples. Thus, we used MS to characterize changes in expression of membrane protein markers before and after short-term induction of osteoblast (OB) differentiation in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and cytochemistry. Quantitative real-time reverse transcription-polymerase chain reaction confirmed the presence of mRNA of these membrane proteins. However, with the exception of ALP, no concordance was detected between the changes in levels of gene and protein expression during OB differentiation. In conclusion, MS-based proteomics can reveal novel markers for MSCs that can be used for their isolation and for monitoring OB differentiation.

Bone Marrow Cells↗

PubMed2004

Increased RANKL/OPG mRNA ratio in iliac bone biopsies from women with hip fractures.

RANKL (receptor activator of NF-kappaB) is a potent physiological inducer of osteoclastogenesis. Its actions are blocked by the decoy receptor osteoprotegerin (OPG), and treatment with OPG blocks bone resorption in postmenopausal women. Both positive and negative associations between serum OPG and bone mineral density (BMD) have been reported in the literature. We hypothesized that decreased OPG production relative to RANKL within bone itself could lead to increased risk of osteoporotic fracture. We included ten women with hip fracture (age 76.3 +/- 8.0 years, N.S, : hip BMD 0.686 +/- 1.3 g/cm2, P < 0.05) and 24 women with osteoarthrosis of the hip (age 72.8 +/- 7.2 years, hip BMD 0.832 +/- 1.1 g/cm(2)). Transiliac biopsies were obtained at the time of surgery. Total RNA was extracted from biopsies and reverse-transcribed. Real-time quantification of mRNA was performed with a SYBR Green I real time PCR assay, calculating relative gene expression with normalization of results for beta actin mRNA. Actin normalized mRNA levels for OPG and interleukin (IL)-6 were significantly lower in fracture patients, with a significantly higher RANKL/OPG ratio in patients with fractures. There was no significant difference in tumor necrosis factor (TNF), IL-1, IL-1ra, or IL-7 expression. IL-6 mRNA levels were lower in fracture patients (P < 0.05). The effect of increased RANKL/OPG ratio (Z = 2.08, P < 0.05) on fracture risk was additive to that of hip BMD T score (Z = -1.95, P < 0.05) when assessed using logistic regression. Elderly women with hip fractures exhibit an increased RANKL/OPG mRNA content of iliac bone. This is associated with increased fracture susceptibility, which is not in itself explained by low BMD.

Absorptiometry, Photon↗